This is generally in the field of biologically active nucleic acid molecules, such as external guide sequences, ribozymes, antisense RNA, and triple helix-forming RNA, and specifically in the area of methods for the identification of sites in target RNA that are accessible to such biologically nucleic acid molecules.
Ribonucleic acid (RNA) molecules can serve not only as carriers of genetic information, for example, genomic retroviral RNA and messenger RNA (mRNA) molecules and as structures essential for protein synthesis, for example, transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules, but also as enzymes which specifically cleave nucleic acid molecules. Such catalytic RNA molecules are called ribozymes.
Drs. Altman and Cech were awarded the Nobel prize in 1989 for the discovery of catalytic RNA. This discovery has generated much interest in commercial applications of ribozymes, particularly in therapeutics (Altman, Proc. Natl. Acad. Sci. USA 90:10898-10900 (1993); Symons, Annu. Rev. Biochem. 61:641-671 (1992); Rossi et al., Antisense Res. Dev., 1:285-288 (1991); Cech, Annu. Rev. Biochem. 59:543-568, (1990)). Several classes of catalytic RNAs (ribozymes) have been described, including intron-derived ribozymes (WO 88/04300; see also, Cech, Annu. Rev. Biochem., 59:543-568, (1990)), hammerhead ribozymes (WO 89/05852 and EP 321021 by GeneShears), hairpin ribozymes (U.S. Pat. No. 5,527,895 to Hampel et al.), and axehead ribozymes (WO 91/04319 and WO 91/04324 by Innovir). Analogues of hammerhead ribozymes useful for specific cleavage of RNA molecules are described in U.S. Pat. No. 5,334,711. Oligomers based on hammerhead ribozymes in which the oligomer and the target RNA each contribute part of the catalytic core are described in WO 97/18312.
Another class of ribozymes includes the RNA portion of an enzyme, RNAse P, which is involved in the processing of transfer RNA (tRNA), a common cellular component of the protein synthesis machinery. Bacterial RNAse P includes two components, a protein (C5) and an RNA (M1). Sidney Altman and his coworkers demonstrated that the M1 RNA is capable of functioning just like the complete enzyme, showing that in Escherichia coli the RNA is essentially the catalytic component, (Guerrier-Takada et al., Cell 35:849-857 (1983)). In subsequent work, Dr. Altman and colleagues developed a method for converting virtually any RNA sequence into a substrate for bacterial RNAse P by using an external guide sequence (EGS), having at its 5' terminus at least seven nucleotides complementary to the nucleotides 3' to the cleavage site in the RNA to be cleaved and at its 5' terminus the nucleotides NCCA (N is any nucleotide)(WO 92/03566 by Yale University, U.S. Pat. No. 5,168,053, and Forster and Altman, Science 238:407-409 (1990)). Using similar principles, EGS/RNAse P-directed cleavage of RNA has been developed for use in eukaryotic systems, (Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); U.S. Pat. No. 5,624,824; WO 95/24489 by Yale University). A short form of eukaryotic external guide sequence has also been described (WO 97/33991 by Innovir Laboratories, Inc.). As used herein, "external guide sequence" and "EGS" refer to any oligonucleotide that forms an active cleavage site for RNAse P in a target RNA.
Although ribozymes theoretically can cleave any desired site in an RNA molecule, in reality not all sites are efficiently cleaved by ribozymes designed or targeted to cleave them. This is especially true in vivo where numerous examples have been described of sites that are inefficiently cleaved by targeted ribozymes. The problem is not a total lack of sites in an RNA molecule of interest, but rather determining which sites, among the many possible sites, can be cleaved most efficiently. This is important since it is often desirable to identify the most efficient sites of cleavage and not just any site that can be cleaved. The process of targeting one or a few sites on an RNA molecule essentially at random and then testing for cleavage is not likely to identify the most efficient sites. Comprehensive testing of all sites is not practical because of the amount of labor involved in making and testing each ribozyme or external guide sequence. WO 96/21731 by Innovir describes selection of efficiently cleaved sites in this manner by making and testing 80 different external guide sequences targeted to different sites. However, this represented only a fraction of the possible sites. Techniques for identifying sites that accessible for cleavage are described in U.S. Pat. No. 5,525,468 and U.S. Pat. No. 5,496,698.
Kawasaki et al., Nucl. Acids Res. 24(15):3010-3016 (1996), describes the use of a transcript encoding a fusion between adenovirus E1A-associated 300 kDa protein (p300) and luciferase to assess the efficiency with which sites in the p300 RNA are cleaved by hammerhead ribozymes in vivo. A few hammerhead ribozymes targeted to sites having GUX triplets (which are required for cleavage by a hammerhead ribozyme) were designed and expressed from a vector in cells. A separate vector expressed the p300-luciferase fusion RNA. Cleavage of sites in the p300 portion of the transcript was assessed by measuring luciferase activity. Kawasaki et al. tested each ribozyme separately.
As an alternative to testing for cleavable sites, or preliminary to such testing, attempts have also been made to predict which sites will be accessible from theoretical considerations or by empirically testing the presence or absence of secondary or tertiary structure at sites in RNA molecules. For example, Ruffner et al., Biochemistry 29:10695-10702 (1990), Zoumadakis and Tabler, Nucl. Acids Res. 23:1192-1196 (1995), Shimayama et al., Biochemistry 34:3649-3654 (1995), Haseloff and Gerlach, Nature 334:585-591 (1988), and Lieber and Strauss, Mol. Cell. Biol. 8:466-472 (1995), describe attempts to use rules of structure formation in RNA to predict cleavable sites. However, the structure of RNA molecules cannot be accurately predicted from theoretical considerations and the determination of actual secondary and tertiary structure of an RNA molecule requires extensive experimentation. Such determinations are often of marginal value since structural determinations are carried out in vitro while the in vivo structure may be different. Accordingly, it would be useful to have a method of determining which sites in an RNA molecule can be efficiently cleaved in vivo. For example, it would be useful to have a method of determining which ribozymes or external guide sequences are most efficient at cleaving or mediating cleavage of an RNA molecule in vivo.
It can also be difficult to identify ribozymes and other biologically active molecules that will function inside cells since not all such biologically active molecules that are functional in vitro are functional in cells because they are, for example, improperly localized, sequestered, or bound by intracellular proteins.
Therefore, it is an object of the present invention to provide a method and compositions for identifying biologically active RNA molecules, such as ribozymes, external guide sequences for ribozymes, antisense RNA, and triple helix-forming RNA, that alter expression of a target RNA molecule most efficiently in vivo.
It is a further object of the present invention to provide a method and compositions for identifying sites in a target RNA, or nucleic acid involved in expression of a target RNA, that are most accessible as target sites for alteration of expression in vivo.
It is a further object of the present invention to provide inhibitory oligomers targeted to sites identified as accessible.